The Ovary of
Tubifex tubifex
(Clitellata,
Naididae, Tubificinae) Is Composed of One,
Huge Germ-Line Cyst that Is Enriched with
Cytoskeletal Components
Anna Z. Urbisz*,Łukasz Chajec, PiotrŚwiątek
Department of Animal Histology and Embryology, University of Silesia, Bankowa 9, 40–007 Katowice, Poland
*anna.urbisz@us.edu.pl
Abstract
Recent studies on the ovary organization and oogenesis in Tubificinae have revealed that their ovaries are small polarized structures that are composed of germ cells in subsequent stages of oogenesis that are associated with somatic cells. In syncytial cysts, as a rule, each germ cell is connected to the central cytoplasmic mass, the cytophore, via only one stable intercellular bridge (ring canal). In this paper we present detailed data about the com-position of germ-line cysts inTubifex tubifexwith special emphasis on the occurrence and distribution of the cytoskeletal elements. Using fixed material and live cell imaging tech-niques, we found that the entire ovary ofT.tubifexis composed of only one, huge multicellu-lar germ-line cyst, which may contain up to 2,600 cells. Its architecture is broadly simimulticellu-lar to the cysts that are found in other clitellate annelids, i.e. a common, anuclear cytoplasmic mass in the center of the cyst and germ cells that are connected to it via intercellular bridges. The cytophore in theT.tubifexcyst extends along the long axis of the ovary in the form of elongated and branched cytoplasmic strands. Rhodamine-coupled phalloidin staining re-vealed that the prominent strands of actin filaments occur inside the cytophore. Similar to the cytophore, F-actin strands are branched and they are especially well developed in the middle and outermost parts of the ovary. Microfilaments are also present in the ring canals that connect the germ cells with the cytophore in the narrow end of the ovary. Using Tubulin-Tracker, we found that the microtubules form a prominent network of loosely and evenly dis-tributed tubules inside the cytophore as well as in every germ cell. The well-developed cytoskeletal elements inT.tubifexovary seem to ensure the integrity of such a huge germ-line cyst of complex (germ cells - ring canals - cytophore) organization. A comparison be-tween the cysts that are described here and other well-known female germ-line cysts is also made.
OPEN ACCESS
Citation:Urbisz AZ, ChajecŁ,Świątek P (2015) The Ovary ofTubifex tubifex(Clitellata, Naididae, Tubificinae) Is Composed of One, Huge Germ-Line Cyst that Is Enriched with Cytoskeletal Components. PLoS ONE 10(5): e0126173. doi:10.1371/journal. pone.0126173
Academic Editor:Claude Prigent, Institut de Génétique et Développement de Rennes, FRANCE
Received:December 19, 2014
Accepted:March 30, 2015
Published:May 22, 2015
Copyright:© 2015 Urbisz et al. This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement:All relevant data are within the paper.
Funding:The authors have no support or funding to report.
Introduction
The formation of germ-line cysts (clusters, nests, clones) seems to be a conserved phase of ga-metogenesis in most invertebrate and vertebrate animals [1–3]. Usually during early oogenesis, germ-line stem cells (GSCs) divide asymmetrically and produce new GSCs and cyst progenitor cells (cystoblasts, Cbs). Then the Cbs divide mitotically several times without full cytokinesis, and as a result, sibling germ cells (cystocytes) are interconnected by broad cytoplasmic chan-nels (stable intercellular bridges, ring canals) and form syncytia [1,2,4]. When a given cyst is completed there are two modes for its future development—panoistic (e.g., some vertebrates such as Xenopus laevisand Mus musculus) in which cytokinesis is completed quickly and each germ cell has the potential to become an oocyte, or meroistic (e.g., some insects such as Dro-sophila melanogaster) in which germ cells are interconnected for a longer period of time and their fates diverge, i.e. usually only a small number (sometimes one) of the germ cells differenti-ate into oocytes while the rest of the cells play the role of supporting cells (nurse cells, tropho-cytes) and finally die [1–8]. It should be added here that in some animals, e.g. in basal insects, female germ-line cysts have never been found [5] and recent studies of the firebratThermobia domesticahave confirmed that individual Cbs develop directly into oocytes (panoistic oogene-sis) in this species [9].
The female germ-line cysts in different animal taxa show a great deal of diversity. The main differences are the number of interconnected cells and the spatial pattern of cell
distribution–the cyst architecture. The simplest, two-celled cysts have been found in some in-sects such asForficula auricularia[10] and in the polychaete annelidOphryotrocha labronica
(Table 1) [11,12], 16-celled cysts are known from the ovaries ofDrosophila melanogaster[8] andXenopus laevis[13], whereas as many as 250 cells have been found in the female cyst in the Strepsiptera [14,15].
As for the architecture of cysts, the cells in a cyst may form linear/almost linear chains–all of the cystocytes except the terminal ones are connected to their sister cells by two ring canals; the terminal cystocytes only have one ring canal [8]. Such linearly organized cysts have been found, e.g., in the polychaete annelidDiopatra cuprea[16] and in some insects such as collem-bolans and mayflies [5,17]. A more complicated pattern of cell distribution is observed in branched cysts, i.e. where the cystocytes are connected to their neighbours via more than two intercellular bridges and form“branches”[4,5,6,8,13,18–20]. Branched cysts have been found and described in detail in model organisms such asDrosophila melanogasterand Xeno-pus laevis(seeTable 1for some details and references).
The organization of the female germ-line cysts in such taxa as clitellate annelids, nematodes and mites is more complicated; the center of the cyst is occupied by an anuclear cytoplasmic mass (cytoplasmic core, central core) of a different shape and size, which is called a cytophore in clitellate annelids, a gonad core or a rachis in nematodes and a medulla in mites, while the germ cells are located at the periphery of the cyst. Additionally, as a rule, each germ cell is con-nected to the cytoplasmic core via only one stable intercellular bridge [21–32]. The number of germ cells in cysts that have a central core is usually high (the lowest known number is 16 in the white worm,Enchytraeus albidus[33]) and usually varies between taxa and may even vary between different germ-cell clusters in the same ovary (e.g., there are from 24 to 44 cells in fish leeches,Piscicola[34]). There are about 1,300 female germ cells that are connected to a long, tube-like central core in the gonads of the nematodeCaenorhabditis elegans(Table 1) [22].
Table 1. The summary of germ-line cyst organization in different groups of animals in conjunction with the cytoskeleton.
Species Cyst geometry Number of germ cells in a cyst
Interconnection of cysts components
Molecular composition of intercellular bridges
Actin cytoskeleton Microtubular cytoskeleton
Tubifex tubifex Cone-like, evidently polarized; long and branched cytophore ([27] this study)
~2000 (including ~ 8 growing oocytes) [this study]
Each germ cell has one ring canal connecting it to the cytophore ([27] this study)
F-actin [this study] Subcortical actin bundles in all germ cells; extensive, long and branched actin bundles within the cytophore reaching intercellular bridge of each germ cell [this study]
Prominent network of microtubules in cytoplasm and around nuclei of nurse cells and oocytes; network of microtubules and parallel bundles within the cytophore [this study]
Drosophila melanogaster
During formation rosette-like with maximally branched pattern of cell interconnections, thenflat or lens-shaped [8,56]
16 (15 nurse cells and 1 oocyte) [4,8,56]
Two germ cells with 4 ring canals, 2 with 3, 4 with 2, and 8 cystocytes with 1 ring canal [8,56]
completely assembled: F-actin, Ov-htsRC, Kelch, Cheerio, Cullin-3, Pav-Klp,Tec29, Src64B, Mucin-D, pTyr proteins, Visgun; temporarily: Cindr, Anilin [4,56]
Subcortical and cytoplasmic F-actin; baskets of F-actin bundles at the ring canals on the nurse cells side during selective transport; the cage of radially arranged F-actin around the nurse cells nuclei to prevent entering their nuclei into the oocyte [56]
Microtubular anterior-posterior gradient of diminishing abundance and polarization within oocyte causing proper localization of
macromolecules and the nucleus; subcortical parallel arrays of microtubules in the oocyte which mix nurse cell cytoplasm entering to the oocyte during the rapid transfer [54,55,63]
Xenopus laevis Rosette-like with maximally branched pattern of cell interconnections [13]
16 (all cells develop as oocytes) [13]
Two germ cells with 4 ring canals, 2 with 3, 4 with 2 and 8 cystocytes with 1 ring canal [13]
F-actin, Kelch, Ov-htsRC [13]
Rich subcortical and radially spread actin-based cytoskeleton in fully grown oocytes [13,64]
Polarized microtubules involved in transport of synchronizing factors in early germ cells [13,64]
Ophryotrocha labronica
Two-celled cysts forming by fragmentation of parental cysts with no consistent pattern of branching (linear or more complex) [12]
2 (1 nurse cell and 1 oocyte) [11,12]
One ring canal
connecting nurse cell with oocyte [11,12]
pTyr proteins [11] Actinfilaments localized in the nurse cell cortex causing a contraction of this cell and allow transferring cytoplasm into the oocyte [11]
Extensive microtubule cytoskeleton in the ring canal of developing cysts forming a scaffold which prevent inappropriate mixing of cytoplasm [11]
Caenorhabditis elegans
U-shaped and polarized with syncytial part (mitotic germ cells) and cellularized growing oocytes; long unbranched cytoplasmic core (rachis) extend through the syncytial part of cyst and disappears within the part of developing oocytes [22,
23,43,44]
~1300 syncytial germ cells and 10–14 oocytes (at a time) [22]
Each germ cells possess one intercellular bridge connecting it to the rachis [22,23,43,44]
Anilin protein ANI-2 [45,46]
Randomly oriented
filaments within the rachis in the pachytene region of the gonad; rich actin
filaments around oocytes nuclei;fine
filaments within the rachis and numerous within ring canals of enlarging oocytes in the direction of cytoplasmic streaming [47]
Baskets of microtubules surround germ cells nuclei from the pachytene region throughout subsequent stages of oogenesis until the oocyte cellularization. Microtubules extend through intercellular bridges and within the rachis [47]
Stomaphis quercus
During formation each ovariole consists of single spherical cyst with germ cells arranged into a rosette; then cyst elongates and forms a centre occupied by more or less long, common cytoplasmic area (trophic core); the anterior part of cyst (tropharium) contains nurse cells; posterior part (vitellarium) contains growing oocytes [48]
32 germ cells in viviparous generations (including 1–2 oocytes); 45–60 in oviparous (including 1 oocyte) [48]
Germ cells connected with trophic core by cytoplasmic bridges; Nurse cells form processes which extend into the trophic core; Vitellogenic oocytes are connected to the trophic core by the broad nutritive cords (modified intercellular bridges) [48]
– – Bundles of microtubules
filling the trophic core; Parallel-arranged microtubules within nutritive cord [48]
No available data (-).
Clitellata in which germ-line clusters have drawn special attention due to their complicated form is Tubificinae. The morphology of the ovary and the course of oogenesis in three repre-sentatives of this group have recently been studied [27]. However, there is still a lack of answers to basic questions such as how many germ-line cysts occur in the entire tubificinin ovary and how many germ cells constitute a single cyst? What is the exact cyst architecture? What is the organization of the microtubular and F-actin cytoskeleton? In the paper presented here, we focus on these details of the germ-line cysts in the sludge wormTubifex tubifex(Tubificinae). In order to address these questions, we analyzed both sectioned and whole-mountedT.tubifex
ovaries using a chemically fixed material and live cell imaging techniques. We found that the entire ovary is composed of only one, huge multicellular (~2,000 cells) germ-line cyst that is en-riched with cytoskeletal elements.
Materials and Methods
Animal material
Specimens ofTubifex tubifex(Müller, 1774) were purchased from commercial sources and maintained in a 10,000 cm3aquarium under laboratory conditions. Only mature specimens with a fully developed clitellum were selected for the study. About 100 specimens were used for all of the analyses.
Differential interference contrast
The specimens ofT.tubifexwere fixed in 4% formaldehyde (freshly prepared from paraformal-dehyde) in PBS (phosphate buffered saline, NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4 8 mM; KH2PO4, 1.5 mM, pH 7.4) for 30–40 min at room temperature and washed in PBS. After that the ovaries were dissected, they were whole-mounted onto microscope slides, and observed under an Olympus BX60 microscope equipped with Nomarski differential interference contrast.
Light microscopy
The individuals ofT.tubifexwere initially fixed with 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4), then isolated segments with gonads were fixed in 2.5% glutaraldehyde in the same buffer at room temperature for several days. After washing in a phosphate buffer, the ma-terial was postfixed for 2 h in 1% OsO4in the same buffer, dehydrated in a graded series of eth-anol that was replaced by acetone and embedded in Epon 812 (Fullam Inc., Latham, NY, USA). Semi-thin sections (0.8μm thick) were stained with methylene blue and examined
under an Olympus BX60 microscope equipped with a DP12 digital camera and AnaliSIS 3.2 (Soft Imaging System) software.
DAPI and propidium iodide staining
After initial fixation in 4% formaldehyde (freshly prepared from paraformaldehyde) in PBS, the individuals ofT.tubifexwere dissected and the ovaries that were obtained were fixed in 4% formaldehyde (for 40 min at room temperature), washed in PBS, dehydrated in a graded etha-nol series, infiltrated and embedded in Histocryl resin (London Resin Company Ltd, Basing-stoke, Hampshire, England). The Histocryl sections (1μm) were double stained with DAPI
(1μg/ml) and propidium iodide (1μg/ml; Sigma) at a ratio 1:1 for 30 min in darkness. Sections
Detection of F-actin
Dissected gonads ofT.tubifexwere fixed in 4% formaldehyde (freshly prepared from parafor-maldehyde) in PBS for 30–40 min at room temperature and stained with rhodamine-conjugat-ed phalloidin (2μg/ml, Sigma) for 40 min in darkness, washed again in PBS and additionally
stained with DAPI (1μg/ml) for 30 min in darkness. Stained ovaries were whole-mounted
onto microscope slides and analyzed under both an Olympus BX60 epifluorescence micro-scope equipped with appropriate filters and an Olympus FV1000 confocal micromicro-scope.
Detection of Microtubules
Sludge worms were anesthetized in ethanol, then dissected in DPBS (Dulbecco’s Phosphate Buffered Saline, Sigma-Aldrich D4031) and incubated in a prewarmed staining solution of Tu-bulin Tracker Green (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C (the TuTu-bulin Tracker solution was prepared according to manufacturer’s protocols). In addition Hoechst 33342 (1μg/ml, Molecular Probes) was added to the staining solution, in order to counterstain
the nuclei. After rinsing with DPBS, the ovaries were whole-mounted onto microscope slides and analyzed under an Olympus BX60 epifluorescence microscope.
Counting the germ cells in the ovary
To count the germ cells, the specimens that were initially fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) in PBS were dissected and the ovaries that were obtained were additionally fixed in the same fixative for 30–40 min at room temperature and stained with DAPI for 30 min in darkness. The ovaries were then whole-mounted onto microscope slides and analyzed under an Olympus FV1000 confocal microscope. The nuclei of the germ cells were counted using ImageJ Software. Ten ovaries were analyzed for the cell count.
Results
General organization of the germ-line cyst in conjunction with the actin filaments A detailed description ofT.tubifexovaries was presented in our earlier paper [27]. The schematic illustration ofT.tubifexovary organization is presented inFig 1. Briefly, the ovary of
T.tubifexis a small (~2mm long), conically shaped and clearly polarized structure with its nar-row end equipped with a thin ligament that connects it to the intersegmental septum (Figs1, 2Aand3). The gradient of germ cell development is observed along the long ovary axis, i.e. the oogonia occur just below the ligament–zone I, below that there are undifferentiated germ cells (cystocytes)–zone II, and germ cells that have two morphologically distinct categories occupy the rest of the ovary–zone III (Figs1–4). We regard these two morphologically different types of germ cells as nurse cells and oocytes (for ultrastructural details and discussion about possible ovary meroism inT.Tubifexsee [27]). Growing oocytes, as a rule, are situated on the surface of the gonad and are arranged on only one side of the ovary (Figs1,2,3,4A and 4B). Somatic cells accompany germ cells both inside the ovary and around the entire ovary and form a thin, outer envelope.
actin filaments within the cytophore (Figs3–5). In zones I and II, where the cytophore has the form of very thin, inconspicuous strands of cytoplasm (initial cytophore; for ultrastructural de-tails see [27]), the bundles of actin filaments are thin (Figs3,5A and 5B). In zone III, where the cytophore becomes more voluminous and occupies the central position in the cyst in the form of a long, broad and branched cytoplasmic core, an actin filaments form prominent cables (Figs2B and 2C–4). Actin cables are even visible as lighter areas inside the cytophore cyto-plasm in the semi-thin sections (Fig 2B and 2C). In zone III the cytophore forms more and more finer branches, in which very thin F-actin bundles occur, and they seem to reach individ-ual germ cells (Figs2B and 2C,5). All of the germ cells in the ovary are connected to the
Fig 1. Schematic illustration of the spatial organization of theT.tubifexovary.The ovary is conically shaped with its narrow end attached to the intersegmental septum (is) via a thin ligament (li) and is polarized along its long axis. Germ cells at subsequent stages of oogenesis occur in three zones (I, II, III): zone I contains oogonia (oo), undifferentiated germ cells (cystocytes) (gc) occur in zone II and germ cells that are morphologically differentiated into growing oocytes (o) and smaller nurse cells (nc) occur in zone III. Oocytes grow on only one side of the ovary and gradually protrude into the body cavity. The entire ovary is composed of only one, huge germ-line cyst. Each germ cell in a cyst (ovary) is connected to the central anuclear core (cytophore) via one stable intercellular bridge (ring canal–ellipse). The cytophore (cy) is poorly developed in zones I and II, whereas it is prominent and branched in zone III. In addition to the germ cells, somatic cells (sc) occur–they are localized inside the ovary and on its surface and form a thin ovary envelope.
cytophore (Fig 5) [27]. The cell count revealed that the total number of germ cells in a cyst (ovary) varies from 1,442 to 2,649 (Table 2); the average number of cells per cyst is 2,040 (N = 10 ovaries). Four to 18 oocytes (on average 8) grow and protrude on the ovary surface (Table 2, Figs2A,3,4A and 4B). The number of germ cells in a given cyst is not constant, be-cause mitotic divisions of germ cells have been found even in fully developed ovaries (Fig 6G).
Each germ cell united into a syncytial germ-line cyst has only one ring canal that connects it to the cytophore (Fig 5B–5E) [27]. Recent ultrastructural studies revealed that the walls of ring canals have a layer of fibrous electron-dense material [27]; the present studies showed that they are enriched with actin filaments (Fig 5). The F-actin was easily detectable in the ring canal walls of oogonia and undifferentiated germ cells (zones I and II), while the F-actin labeling was much weaker in the ring canals of the nurse cells and oocytes in zone III (Figs3–5). Actin fila-ments are also distributed in the cortical cytoplasm in both individual germ cells and the cyto-phore (Figs4,5A, 5B, 5D and 5E).
Fig 2. General organization of an ovarian cyst inT.tubifex.(A) The ovary ofTubifex tubifexis conically shaped and polarized. Three zones can be distinguished (I, II and III). Each zone contains germ cells at subsequent stages of oogenesis: oogonia in zone I, undifferentiated cells in zone II and nurse cells and oocytes in zone III. In the third zone, several oocytes (o) grow successively on only one side of the ovary and protrude into the body cavity. An extensive cytophore (cy) is marked in this zone. Whole-mounted
preparation, Nomarski interference contrast. (B) and (C) sections through zone III, in which two morphologically distinct germ cell categories occur–nurse cells (nc) and oocytes (o). The branched cytophore (cy) occupies the central position in the germ-line cyst. Arrowheads mark the cytophore regions that are enriched with cytoskeletal elements (see Figs2Aand3). Arrows point to the somatic cells that accompany the germ cells inside the ovary and form the outer ovary envelope; nu–oocyte nucleus. B longitudinal section, fluorescence microscopy, Histocryl semi-thin section double stained with DAPI and PI; C cross section, light microscopy, Epon semi-thin section stained with methylene blue.
Localization of microtubules
We found numerous microtubules in the germ-line cyst inT.tubifexovaries using Tubulin Tracker Green. The bundles of microtubules are distributed in both the cytoplasm of the germ cells and in the cytophore (Fig 6A–6D and 6F). Microtubules form a dense network that fills the entire cytoplasm and encircles the nuclei in the germ cells (Fig 6A–6D and 6F). Similarly, a prominent network of microtubules was also seen in the cytoplasm of vitellogenic oocytes, which lose contact with the cytophore and float freely in the coelomic fluid (Fig 6E). Microtu-bules were observed filling the whole volume of the cytophore, and they even extended out into its smaller branches (Fig 6A-6D). Microtubules were also detected inside the ring canals that connect the germ cells with the cytophore (Fig 6D).
As in other clitellate annelids, the germ cells in theT.tubifexovary are associated with so-matic cells (Fig 1,2B, 2Cand6H) [27]. Somatic cells are localized both inside the germ-line cyst–between the germ cells and the cytophore and on the surface of the cyst/ovary where they form a thin envelope. The somatic cells are easily distinguishable from the germ cells; they are always smaller than the germ cells, and extremely elongated with long cytoplasmic projec-tions. The nuclei of the somatic cells are small and flattened with condensed chromatin, which gives a strong signal after staining with DAPI or Hoechst (Figs1,2B, 2Cand6H). In the cyto-plasm of somatic cells the microtubules were also observed, especially the bundles of microtu-bules are localized along the long cytoplasmatic projections of these cells (Fig 6H).
Discussion
The entire ovary ofT.tubifexis composed of only one germ-line cyst that has an architecture that is similar to other clitellate annelids
The present study allowed us to analyze the ovary organization inT.tubifex. We found that only one multicellular germ-line cyst constitutes the entire ovary of this species and that this cyst has one long, branched cytophore that extends along the long ovary axis. TheT.tubifex
ovarian cyst is polarized, and therefore the process of oogenesis occurs along this axis. The number of germ cells in a single cyst (= ovary) is nearly 2,000 and on average eight oocytes grow at the same time (seeTable 2). However, the cell number in the cysts is not constant due to the mitotic divisions of the germ cells, which may even occur in fully developed ovaries that have growing oocytes. A similar asynchrony in germ cell divisions has been observed in some other clitellate groups i.e. [26,30,35,36], which indicates that germ cells can still proliferate even in ovaries in which vitellogenic oocytes are already formed.
Studies of ovary organization and oogenesis in clitellate annelids have shown that the for-mation of germ-line cysts is a conserved phase of gametogenesis. In all of the clitellate annelids that have been studied to date, as a rule, each germ cell is connected to the common cyto-plasmic mass (cytophore) by only one stable cytocyto-plasmic bridge [24–28,30,31,33–38]. Despite that general rule, the cyst morphology varies substantially between different clitellate taxa with respect to the number of germ cells, the level of cytophore development and the ratio of nurse cells to oocytes. In the majority of clitellate annelids (Tubificinae, Lumbricida, Lumbriculida, Branchiobdellida, Acanthobdellida, Glossiphoniidae, Erpobdelliformes and Hirudiniformes)
Fig 3. The cytophore organization in an ovarian cyst ofT.tubifex.The entire ovary was stained with DAPI for the visualization of nuclei (A) and rhodamine-conjugated phalloidin to detect F-actin (B); A merged view (C). Maximum-intensity projections of the Z stacks that cover the entire ovary indicate that the entire ovary is composed of one multicellular germ-line cyst. A branched cytophore (stars) extends along the long axis of the ovary in the cyst; the cytophore is thin and inconspicuous in zones I and II, while it is more voluminous and branched in zone III; gc–germ cells, o–oocytes, nc–nurse cells. Confocal microscopy.
ovaries are composed of irregularly shaped and multicellular cysts, however, the exact number of cysts in an ovary and germ-cells in a cyst has not yet been determined [24–26,28,31,35,37, 38]. In contrast, spherical cysts that are composed of a low number of cells occur only in the whitewormEnchytraeus albidus(Enchytraeidae) and in fish leeches (Piscicolidae) (seeTable 2) ([33,34,39], our unpublished results).
Recent papers that have been devoted to oogenesis in clitellates annelids describe two cate-gories of germ cells, which differentiate during the process–several cells (rarely one) continue oogenesis, grow considerably and become oocytes, while the majority of germ cells in a given cyst withdraw from meiosis, do not gather nutrients or grow, and do not seem to develop into oocytes (for details seeTable 2). Due to these morphological differences, ovary meroism is sug-gested in clitellate annelids and these two categories of germ cells are usually regarded as oo-cytes and nurse cells, respectively [25–28,30,31,34–38]. In this paper, we also regard the two morphologically distinct cell categories that are found in zone III as nurse cells and oocytes. However, in order to determine whether the ovaries in Clitellata are really meroistic (i.e. the nurse cells supply growing oocytes with macromolecules and cell organelles and eventually die) specifically dedicated studies are needed. To date, the only experimental evidence that
Fig 4. F-actin localization in aT.tubifexovarian cyst.The entire ovary double stained with DAPI and rhodamine-conjugated phalloidin. Maximum-intensity projections of the Z stacks that cover the interior of the ovary. (A and B) whole ovaries, (C) detail of area between zones II and III. Long bundles of actin filaments occur (stars) in the branched cytophore (cy). F-actin is also localized in the cortical cytoplasm (arrows) of nurse cells (nc) and oocytes (o). Confocal microscopy.
doi:10.1371/journal.pone.0126173.g004
Fig 5. Organization of F-actin in an ovarian cyst ofT.tubifex.(A and B) The initial cytophore with thin F-actin bundles occurs at the narrow end of the ovary (zones I and II) (stars). The cytophore with its F-F-actin bundles reaches every germ cell (gc). The ring canals that connect the germ cells to the cytophore have rims that are enriched with F-actin (arrows). The F-actin also occurs in the cortical cytoplasm of the germ cells (arrowheads). Fluorescence microscopy, whole-mounted preparation stained with rhodamine conjugated phalloidin. C-E Fragment of zone II double stained with DAPI (C) and rhodamine-phalloidin (D); merged view (E). The cytophore has thin bundles of F-actin (stars) that reach each germ cell (gc). The ring canals rims are enriched with F-actin (arrows) and cortical F-actin can be observed in the germ cells (arrowheads).
Fluorescence microscopy.
supports ovary meroism in Clitellata comes from old autoradiographic studies of oogenesis in the leechGlossiphonia complanata. These studies showed that because H3-uridine is actively incorporated into the nucleus and nucleolus of nurse cells, and then labeled RNA is passed into the growing oocytes [40].
Female germ-line cysts in
T
.
tubifex
in comparison with other species
Female germ-line cysts have been studied intensively in only a few groups of invertebrates and vertebrates, primarily in the fruit fly,Drosophila melanogaster. Studies of this species provided interesting data, including the molecular and cellular aspects of germ-line cyst development and function, i.e.: the role of germ-line stem cells, the structure of ring canals, the formation and function of the fusome, germ-line cyst polarization and oocyte determination and the or-ganization of the cytoskeleton (Table 1); for details see [3–5,7,8,41,42].
Although female germ-line cysts in the fruit fly are the best known, they are only one specif-ic example of female cyst organization. To show the diversity of female cyst organization in an-imals and for comparative purposes, we chose several others that have been studied in this aspect, species such as the polychaetous annelidOphryotrocha labronica[11,12], the nematode
Caenorhabditis elegans[22,23,43–47], the aphidStomaphis quercus[48] and among verte-brates the frogXenopus laevis[13]. A comparison of several aspects of the composition of fe-male germ-line cysts, such as its spatial organization, mode of interconnections between cyst components, participation of the cytoskeleton in cyst stability and oogenesis betweenT.tubifex
and the above-mentioned species are presented inTable 1.
When we compare the multicellular germ-line cysts ofT.tubifexwith other non-annelid species, the similarity of the cyst architecture in the species studied here and inC.elegansis conspicuous (Table 1). The distal arm and loop of the U-shaped gonad inC.elegansis also composed of a single syncytial cyst. In both cases, the cysts are evidently polarized given that the syncytium contains cells that are in various stages of oogenesis at the same time. InC. ele-gansa common cytoplasmic core (gonad core) occurs in the center of the cyst, runs through the syncytial part of the gonad and each germ cell is connected to it by one intercellular bridge (rachis bridge) [22,23,43–47]. Similarly, as a rule, each germ cell inT.tubifexis also connected to a common cytoplasm by only one stable intercellular bridge. However, in contrast toT. tubi-fex, the rachis inC.elegansis cylinder-like and unbranched. Hirsh and colleagues (1976) stated that about 1,300 mitotic nuclei and 10–14 growing oocytes occur at the same time in the gonad ofC.elegans. We found 2,000 cells per syncytial cyst on average inT.tubifex. The average
Table 2.
Ovary Number of germ cells Number of growing oocytes
1 1905 8
2 1469 0
3 1442 8
4 1752 9
5 2521 13
6 1845 18
7 2400 11
8 2002 5
9 2413 7
10 2649 5
Average 2039,8 8,4
number of oocytes that grow at the same time was eight. As was mentioned, two morphologi-cally distinct germ cell categories regarded as nurse cells and oocytes have been described inT.
tubifexovaries. There are no morphologically distinguishable nurse cells inC.elegans; however, many experimental data suggest ovary meroism. Wolke et al. (2007) showed cytoplasmic streaming within the cyst in theC.elegansgonad. During this process, cytoplasmic materials flow from a region in the distal gonad where transcriptionally active pachytene-stage germ cells are localized into the proximal region towards the growing, transcriptionally inactive oo-cytes. Thus, the pachytene-stage cells can be considered to be a transient nurse cell population [47]. Additionally, it has been demonstrated that about half of theC.elegansgerm line cells die in late pachytene [44], and therefore if these cells supply other germ cells via the gonad core, they function as“true”nurse cells [47].
The germ-line cysts that were found inT.tubifexshow several striking analogous similari-ties with the syncytial cysts that are found in the telotrophic meroistic ovarioles of Hemiptera. However, the germ-line cysts in Hemiptera develop in a significantly different way than those inT.tubifexi.e. the cysts in hemipterans are branched with a prominent fusome [5,48,49], whereas in the developing cysts of clitellate annelids neither branchings nor a fusome were found [50]. In hemipterans, only one germ-line cyst exists in each ovariole (which is in our
Fig 6. Localization of microtubules in aT.tubifexovarian cyst.Zone III. Live cells stained with Hoechst 33342 (A) and Tubulin Tracker Green (B); (C-H) merged. (A-D) Microtubules form a network that fills the entire cytoplasm of the cytophore (cy); note microtubules in the ring canals that connect the germ cells (gc) with the cytophore (arrowheads) and the bundles of microtubules in the cytophore branches (stars in D). (E-F) A prominent network of microtubules (arrows) occurs in the cytoplasm of the germ cells (gc) and in the vitellogenic oocytes (o); nu–nucleus. (G) Mitotic spindles (arrowheads) of dividing germ cells were observed in zone III; stars indicate metaphase plates. (H) Long bundles of microtubules (arrowheads) were also detected in the cytoplasmic projections of the somatic cells (arrows point to the nuclei of these cells); gc–germ cells, o–oocytes. Fluorescence microscopy.
point of view, functionally equivalent to theT.tubifexovary). There are fewer of germ cysts per syncytium in hemipterans (Table 1; e.g. up to 320 inPsylla alni(Psyllinea)) than in the sludge worm [5], although the germ cells are morphologically diversified into nurse cells and oocytes in both cases [27,48,51,52]. The nurse cells in hemipterans occupy the apical zone of the ovar-ioles (the called tropharium), and linearly arranged oocytes usually grow below it (the so-called vitellarium) [5,48,49]. Interestingly, the central part of the tropharium is occupied by a large anuclear cytoplasmic area (the trophic core) and each germ cell in a cyst is connected to it. The trophic core is usually filled with bundles of microtubules [48,51,52]. Numerous nurse cells in the syncytial cysts inT.tubifexare equivalent to the hemipteran tropharia, whereas the cytophore is the functional equivalent of the trophic core.
Cytoskeleton in syncytial cysts
The active roles of the microfilaments and microtubules during oogenesis have been experi-mentally demonstrated in different species. The cytoskeleton plays an integral role in oogenesis e. g.: in the maintenance of the structural integrity of all of the germ-line cysts; the transport of cell components (such as RNAs, macromolecules and cell organelles) from one germ cell to an-other through intercellular bridges (ring canals); the proper positioning of germ cell nuclei dur-ing this process (for references and details seeTable 1). Two phases of this actin-dependent transport were distinguished inD.melanogaster–the first (slow) included the selective direc-tional transfer through the ring canals into the oocyte, and the second (fast, called dumping or cytoplasmic streaming) in which the nurse cells transfer the remaining content (except for the nuclei) into the growing oocyte [7,53–56]. Actin-dependent transfer has also been described in
C.elegansoogenesis [47] (Table 1).
Our studies revealed that the extremely large syncytial cysts in the sludge worm are enriched with cytoskeletal elements such as F-actin and microtubules. Extensive actin bundles run with-in the cytophore and seem to reach with-individual germ cells. These bundles appear to form a scaf-fold and may play a structural role in the maintenance of a cyst’s integrity. Future experimental studies that are devoted to the transport of cellular components inT.tubifexusing, e.g. injected particle tracking in living cells or toxins that alter the cell cytoskeleton will enable us to find out whether the prominent actin and microtubular cytoskeleton also plays an active role during oogenesis.
As in other germ-line clusters that have been studied [3,57] the germ cells inT.tubifexare interconnected by stable intercellular bridges (ring canals) [27, this study]. Our results in the present study reveal that the ring canals inT.tubifexcontain F-actin. Filamentous actin seems to be a widespread and common component of the ring canals that stabilizes the ring canals after their formation [3,57,58]. However, in some cases, such as in the fully developed male germ cysts ofD.melanogaster, there is no F-actin in the ring canals [59]. Filamentous actin in intercellular bridges has also been recorded in other clitellate annelids such as branchiobdellid
Branchiobdella parasitica[30] and the fish leechPiscicola geometra[34] and in several arthro-pod taxa, including Diptera, Hemiptera, Coleoptera and Branchioarthro-poda [48,57,58,60–62].
Conclusions
Acknowledgments
We are thankful to the Reviewers, whose helpful suggestions have improved our manuscript.
Author Contributions
Conceived and designed the experiments: AZU. Performed the experiments: AZUŁC.
Ana-lyzed the data: AZU PŚ. Contributed reagents/materials/analysis tools: AZUŁC PŚ. Wrote the
paper: AZU PŚ.
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